The Dynamic Plasticity of P. aeruginosa CueR Copper Transcription Factor upon Cofactor and DNA Binding.

Bacteria use specialized proteins, like transcription factors, to rapidly control metal ion balance. CueR is a Gram-negative bacterial copper regulator. The structure of E. coli CueR complexed with Cu(I) and DNA was published, since then many studies have shed light on its function. However, P. aeruginosa CueR, which shows high sequence similarity to E. coli CueR, has been less studied. Here, we applied room-temperature electron paramagnetic resonance (EPR) measurements to explore changes in dynamics of P. aeruginosa CueR in dependency of copper concentrations and interaction with two different DNA promoter regions. We showed that P. aeruginosa CueR is less dynamic than the E. coli CueR protein and exhibits much higher sensitivity to DNA binding as compared to its E. coli CueR homolog. Moreover, a difference in dynamical behavior was observed when P. aeruginosa CueR binds to the copZ2 DNA promoter sequence compared to the mexPQ-opmE promoter sequence. Such dynamical differences may affect the expression levels of CopZ2 and MexPQ-OpmE proteins in P. aeruginosa. Overall, such comparative measurements of protein-DNA complexes derived from different bacterial systems reveal insights about how structural and dynamical differences between two highly homologous proteins lead to quite different DNA sequence-recognition and mechanistic properties.

Refining the Dynamic Network of T2SS Endopilus Tip Heterocomplex Combining cw-EPR and Nitroxide-GdIII Distance Measurements.

The type 2 secretion system (T2SS) is a bacterial nanomachine composed of an inner membrane assembly platform, an outer membrane pore and a dynamic endopilus. T2SS endopili are organized into a homo-multimeric body formed by the major pilin capped by a heterocomplex of four minor pilins. The first model of the T2SS endopilus was recently released, even if structural dynamics insights are still required to decipher the role of each protein in the full tetrameric complex. Here, we applied continuous-wave and pulse EPR spectroscopy using nitroxide-gadolinium orthogonal labelling strategies to investigate the hetero-oligomeric assembly of the minor pilins. Overall, our data are in line with the endopilus model even if they evidenced conformational flexibility and alternative orientations at local scale of specific regions of minor pilins. The integration of different labelling strategies and EPR experiments demonstrates the pertinence of this approach to investigate protein-protein interactions in such multiprotein heterocomplexes.

Electron spin relaxation time of Ni(II) ion in hexapyrazole zinc(II) dinitrate at 300 K.

Understanding the electron spin relaxation properties of paramagnetic species is a fundamental requirement to use them as a probe to measure distances between sites in biomolecules by electron paramagnetic resonance (EPR) spectroscopy. Even though Ni(II) ion is an essential trace element for many species, relaxation properties are not well understood. Herein, the polycrystalline sample of Ni(II) ion magnetically diluted in Zn(Pyrazole)6 (NO3 )2 (Ni/ZPN) has been studied in detail by EPR spectroscopy to explore the electron spin relaxation time. Progressive continuous-wave (CW) EPR power saturation study on Ni/ZPN at 300 K yielded 907 mW as the P1/2 value. The cavity constant (KQ ) has been calculated using tempol in PVA-BA glass matrix and the product of electron spin-lattice relaxation time (T1 ) and spin-spin relaxation time (T2 ) for Ni/ZPN at 300 K has been reported for the first time.

Experimental confirmation of the formation of collective modes of the magnetization motion of paramagnetic particles in dilute solutions due to spin exchange.

Experimental confirmation of the manifestations of new spin exchange paradigm in EPR spectra of 14N nitroxide radical solutions is presented. It was shown that in the region of relatively low concentrations of radicals, the two side components of the spectrum have a mixed shape (the sum of the absorptive line and dispersive line). The dispersion contributions in these two lines have opposite signs. As the concentration of radicals increases, the contribution of dispersion passes through an extremum and in the region of maximum contribution of dispersion, the contribution of absorption to these two lines changes sign. In the region of high concentrations of radicals, when one homogeneously broadened line is practically observed, it turns out that these side components have resonant frequencies that do not coincide with the frequency of the center of gravity of the spectrum.

Characterisation of an unusual cysteine pair in the Rieske carnitine monooxygenase CntA catalytic site.

Rieske monooxygenases undertake complex catalysis integral to marine, terrestrial and human gut-ecosystems. Group-I to -IV Rieske monooxygenases accept aromatic substrates and have well-characterised catalytic mechanisms. Nascent to our understanding are Group-V members catalysing the oxidation/breakdown of quaternary ammonium substrates. Phylogenetic analysis of Group V highlights a cysteine residue-pair adjacent to the mononuclear Fe active site with no established role. Following our elucidation of the carnitine monooxygenase CntA structure, we probed the function of the cysteine pair Cys206/Cys209. Utilising biochemical and biophysical techniques, we found the cysteine residues do not play a structural role nor influence the electron transfer pathway, but rather are used in a nonstoichiometric role to ensure the catalytic iron centre remains in an Fe(II) state.

A comparison of pulse and CW EPR T2-relaxation measurements of an inhomogeneously broadened nitroxide spin probe undergoing Heisenberg spin exchange.

Nitroxide spin probes are inhomogeneously broadened (IHB) by intramolecular hyperfine interactions with protons (deuterons) producing lines of Voigt shape. Thus, to study T2 relaxation by continuous wave (CW) EPR, the Voigt must be deconvoluted to find the Lorentzian component. For homogeneously broadened lines, T2 is obtained directly from the Lorentzian line widths ΔHppL; however, for IHB lines finding T2 from ΔHppL is more complicated. It has been known for many years that values of ΔHppL of high precision may be obtained from IHB lines; however, direct, accurate comparison of spin exchange frequencies obtained from electron spin echo decay and CW EPR data has been lacking. It is demonstrated here that despite complications in the interpretation of experiments, these two techniques yield the same spin exchange rate constant for spin probes that are the most difficult to treat.

Towards high performance dielectric microwave resonators for X-band EPR spectroscopy.

Microwave (MW) resonators in Electron Paramagnetic Resonance (EPR) spectroscopy concentrate the MW magnetic field (B1) at the sample and separate the MW electric field from the sample. There are numerous experimental methods in EPR spectroscopy which all impose different requirements on MW resonators (e.g. high or low quality factor, MW conversion, and B1-field homogeneity). Although commercial spectrometers offer standardized MW resonators for a broad application range, newly emerging and highly-specialized research fields push these spectrometers to or beyond their sensitivity limits. Optimizing the MW resonator offers one direct approach to improve the sensitivity. Here we present three low-cost optimization approaches for a commercially available X-band (9-10 GHz) MW resonator for three experimental purposes (continuous-wave (CW), transient and pulse EPR). We obtain enhanced MW conversion factors for all three optimized resonators and higher quality factors for two optimized resonators. The latter is important for CW and transient EPR. Furthermore, we fabricated a resonator which features an extended area of homogeneous B1-field and, hence, improved pulse EPR performance. Our results demonstrate that small changes to a commercial MW resonator can enhance its performance in general or for specific applications.

The use of EPR spectroscopy to study transcription mechanisms.

Electron paramagnetic resonance (EPR) spectroscopy has become a promising structural biology tool to resolve complex and dynamic biological mechanisms in-vitro and in-cell. Here, we focus on the advantages of continuous wave (CW) and pulsed EPR distance measurements to resolve transcription processes and protein-DNA interaction. The wide range of spin-labeling approaches that can be used to follow structural changes in both protein and DNA render EPR a powerful method to study protein-DNA interactions and structure-function relationships in other macromolecular complexes. EPR-derived data goes well beyond static structural information and thus serves as the method of choice if dynamic insight is needed. Herein, we describe the conceptual details of the theory and the methodology and illustrate the use of EPR to study the protein-DNA interaction of the copper-sensitive transcription factor, CueR.

EPR of copper centers in the prion protein.

Most proteins implicated in neurodegenerative diseases bind metal ions, notably copper and zinc. Metal ion binding may be part of the protein's function or, alternatively, may promote a deleterious gain of function. With regard to Cu2+ ions, electron paramagnetic resonance techniques have proven to be instrumental in determining the biophysical characteristics of the copper binding sites, as well as structural features of the coordinating protein and how they are impacted by metal binding. Here, the most useful methods are described as they apply to the prion protein, which serves as a model for the broader spectrum of neurodegenerative proteins.

Pinholin S21 mutations induce structural topology and conformational changes.

The bacteriophage infection cycle is terminated at a predefined time to release the progeny virions via a robust lytic system composed of holin, endolysin, and spanin proteins. Holin is the timekeeper of this process. Pinholin S21 is a prototype holin of phage Φ21, which determines the timing of host cell lysis through the coordinated efforts of pinholin and antipinholin. However, mutations in pinholin and antipinholin play a significant role in modulating the timing of lysis depending on adverse or favorable growth conditions. Earlier studies have shown that single point mutations of pinholin S21 alter the cell lysis timing, a proxy for pinholin function as lysis is also dependent on other lytic proteins. In this study, continuous wave electron paramagnetic resonance (CW-EPR) power saturation and double electron-electron resonance (DEER) spectroscopic techniques were used to directly probe the effects of mutations on the structure and conformational changes of pinholin S21 that correlate with pinholin function. DEER and CW-EPR power saturation data clearly demonstrate that increased hydrophilicity induced by residue mutations accelerate the externalization of antipinholin transmembrane domain 1 (TMD1), while increased hydrophobicity prevents the externalization of TMD1. This altered hydrophobicity is potentially accelerating or delaying the activation of pinholin S21. It was also found that mutations can influence intra- or intermolecular interactions in this system, which contribute to the activation of pinholin and modulate the cell lysis timing. This could be a novel approach to analyze the mutational effects on other holin systems, as well as any other membrane protein in which mutation directly leads to structural and conformational changes.

Characterization of Paramagnetic Iron-Sulfur Clusters Using Electron Paramagnetic Resonance Spectroscopy.

Continuous-wave (CW) electron paramagnetic resonance (EPR) spectroscopy is a powerful ally in characterizing the multitude of redox-active iron-sulfur cluster-containing ([Fe-S]) species present in biological samples. The technique detects only those clusters that are paramagnetic-having a nonzero total electron spin (S > 0)-thus, it can discriminate between clusters in different oxidation states. The low-temperature CW-EPR spectrum of an [Fe-S] yields the three magnetic g-values that serve as a fingerprint of its electronic structure. This chapter briefly describes the underlying theory that defines this electronic structure and provides a recipe for the acquisition and analysis of EPR spectra of [Fe-S] proteins.

Influence of different polymer belts on lipid properties in nanodiscs characterized by CW EPR spectroscopy.

With this study we aim at comparing the well-known lipid membrane model system of liposomes and polymer-encapsulated nanodiscs regarding their lipid properties. Using differential scanning calorimetry (DSC) and continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy, we characterize the temperature-dependent lipid behavior within 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes and nanodiscs made from such liposomes by application of various polymers based on styrene-co-maleic acid (SMA), diisobutylene-alt-maleic acid (DIBMA), and styrene-co-maleic amide sulfobetaine (SMA-SB), a new SMA-derived copolymer containing sulfobetaine side chains. By incorporating a spin label doxyl moiety into the lipid bilayer in position 16 or 5 we were able to study the micropolarity as well as rotational restrictions onto the lipids in the apolar bilayer center and the chain region adjacent to the carbonyl groups, respectively. Our results suggest that all polymers broaden the main melting transition of DMPC, change the water accessibility within the lipid bilayer, and exhibit additional constraints onto the lipids. Independent of the used polymer, the rotational mobility of both spin-labeled lipids decreased with DIBMA exerting less restraints probably due to its aliphatic side chains. Our findings imply that the choice of the solubilizing polymer has to be considered an important step to form lipid nanodiscs which should be included into research of lipid membranes and membrane proteins in the future.

Cryogenically frozen PEGylated liposomes and micelles: Water penetration and polarity profiles.

Poly(ethylene glycol) (PEG)-grafted lipid dispersions are widely investigated in fundamental and biotechnological research for their successful use in drug-delivery. Here, we consider mixtures of the bilayer-forming lipid dipalmitoylphosphatidylcholine (DPPC) with the micelle-forming lipid PEG:2000-phosphatidilethanolamine (PEG:2000-DPPE) fully hydrated in D2O and measured at 77 K. Electron Spin Echo Envelope Modulation and continuous wave Electron Paramagnetic Resonance of chain-labelled lipids are employed to detect the extent of solvent permeation and the environmental polarity, respectively, across the hydrocarbon regions of the lipid assemblies. Sigmoidal water penetration and polarity profiles are described in sterically stabilized liposomes (SSL) formed at submicellar content of PEG:2000-DPPE incorporated in DPPC. Compared to DPPC bilayers, SSL show increased hydrophobicity at both the polar/apolar interface and the chain termini, and a broader transition that is shifted toward the interface. Solvent exposure and polarity decrease on going down the chain in PEG:2000-DPPE micelles. However, compared to SSL, polymer-lipid micelles show higher solvent permeation at any chain segment and the chain termini are accessible to water. In any sample, heterogeneity is found in H-bond formation between the spin-label nitroxide groups and the solvent molecules. The results at cryogenic temperature add new insights into the biophysico-chemical characterization of PEGylated lipid dispersions.

Structural characterization of styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) using EPR spectroscopy.

Spectroscopic studies of membrane proteins (MPs) are challenging due to difficulties in preparing homogenous and functional lipid membrane mimetic systems into which membrane proteins can properly fold and function. It has recently been shown that styrene-maleic acid (SMA) copolymers act as a macromolecular surfactant and therefore facilitate the formation of disk-shaped lipid bilayer nanoparticles (styrene-maleic acid copolymer-lipid nanoparticles (SMALPs)) that retain structural characteristics of native lipid membranes. We have previously reported controlled synthesis of SMA block copolymers using reversible addition-fragmentation chain transfer (RAFT) polymerization, and that alteration of the weight ratio of styrene to maleic acid affects nanoparticle size. RAFT-synthesis offers superior control over SMA polymer architecture compared to conventional radical polymerization techniques used for commercially available SMA. However, the interactions between the lipid bilayer and the solubilized RAFT-synthesized SMA polymer are currently not fully understood. In this study, EPR spectroscopy was used to detect the perturbation on the acyl chain upon introduction of the RAFT-synthesized SMA polymer by attaching PC-based nitroxide spin labels to the 5th, 12th, and 16th positions along the acyl chain of the lipid bilayer. EPR spectra showed high rigidity at the 12th position compared to the other two regions, displaying similar qualities to commercially available polymers synthesized via conventional methods. In addition, central EPR linewidths and correlation time data were obtained that are consistent with previous findings.

CW EPR and DEER Methods to Determine BCL-2 Family Protein Structure and Interactions: Application of Site-Directed Spin Labeling to BAK Apoptotic Pores.

The continuous wave (CW) and pulse electron paramagnetic resonance (EPR) methods enable the measurement of distances between spin-labeled residues in biopolymers including proteins, providing structural information. Here we describe the CW EPR deconvolution/convolution method and the four-pulse double electron-electron resonance (DEER) approach for distance determination, which were applied to elucidate the organization of the BAK apoptotic pores formed in the lipid bilayers.

Assessing topology and surface orientation of an antimicrobial peptide magainin 2 using mechanically aligned bilayers and electron paramagnetic resonance spectroscopy.

Aligned CW-EPR membrane protein samples provide additional topology interactions that are absent from conventional randomly dispersed samples. These samples are aptly suited to studying antimicrobial peptides because of their dynamic peripheral topology. In this study, four consecutive substitutions of the model antimicrobial peptide magainin 2 were synthesized and labeled with the rigid TOAC spin label. The results revealed the helical tilts to be 66°â€¯±â€¯5°, 76°â€¯±â€¯5°, 70°â€¯±â€¯5°, and 72°â€¯±â€¯5° for the TOAC substitutions H7, S8, A9, and K10 respectively. These results are consistent with previously published literature. Using the EPR (electron paramagnetic resonance) mechanical alignment technique, these substitutions were used to critically assess the topology and surface orientation of the peptide with respect to the membrane. This methodology offers a rapid and simple approach to investigate the structural topology of antimicrobial peptides.

Exploring Structure, Dynamics, and Topology of Nitroxide Spin-Labeled Proteins Using Continuous-Wave Electron Paramagnetic Resonance Spectroscopy.

Structural and dynamical characterization of proteins is of central importance in understanding the mechanisms underlying their biological functions. Site-directed spin labeling (SDSL) combined with continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy has shown the capability of providing this information with site-specific resolution under physiological conditions for proteins of any degree of complexity, including those associated with membranes. This chapter introduces methods commonly employed for SDSL and describes selected CW EPR-based methods that can be applied to (1) map secondary and tertiary protein structure, (2) determine membrane protein topology, (3) measure protein backbone flexibility, and (4) reveal the existence of conformational exchange at equilibrium.

CW dipolar broadening EPR spectroscopy and mechanically aligned bilayers used to measure distance and relative orientation between two TOAC spin labels on an antimicrobial peptide.

An EPR membrane alignment technique was applied to measure distance and relative orientations between two spin labels on a protein oriented along the surface of the membrane. Previously we demonstrated an EPR membrane alignment technique for measuring distances and relative orientations between two spin labels using a dual TOAC-labeled integral transmembrane peptide (M2δ segment of Acetylcholine receptor) as a test system. In this study we further utilized this technique and successfully measured the distance and relative orientations between two spin labels on a membrane peripheral peptide (antimicrobial peptide magainin-2). The TOAC-labeled magainin-2 peptides were mechanically aligned using DMPC lipids on a planar quartz support, and CW-EPR spectra were recorded at specific orientations. Global analysis in combination with rigorous spectral simulation was used to simultaneously analyze data from two different sample orientations for both single- and double-labeled peptides. We measured an internitroxide distance of 15.3Šfrom a dual TOAC-labeled magainin-2 peptide at positions 8 and 14 that closely matches with the 13.3Šdistance obtained from a model of the labeled magainin peptide. In addition, the angles determining the relative orientations of the two nitroxides have been determined, and the results compare favorably with molecular modeling. This study demonstrates the utility of the technique for proteins oriented along the surface of the membrane in addition to the previous results for proteins situated within the membrane bilayer.

Homogeneity and EPR metrics for assessment of regular grids used in CW EPR powder simulations.

CW EPR powder spectra may be approximated numerically using a spherical grid and a Voronoi tessellation-based cubature. For a given spin system, the quality of simulated EPR spectra depends on the grid type, size, and orientation in the molecular frame. In previous work, the grids used in CW EPR powder simulations have been compared mainly from geometric perspective. However, some grids with similar homogeneity degree generate different quality simulated spectra. This paper evaluates the grids from EPR perspective, by defining two metrics depending on the spin system characteristics and the grid Voronoi tessellation. The first metric determines if the grid points are EPR-centred in their Voronoi cells, based on the resonance magnetic field variations inside these cells. The second metric verifies if the adjacent Voronoi cells of the tessellation are EPR-overlapping, by computing the common range of their resonance magnetic field intervals. Beside a series of well known regular grids, the paper investigates a modified ZCW grid and a Fibonacci spherical code, which are new in the context of EPR simulations. For the investigated grids, the EPR metrics bring more information than the homogeneity quantities and are better related to the grids' EPR behaviour, for different spin system symmetries. The metrics' efficiency and limits are finally verified for grids generated from the initial ones, by using the original or magnetic field-constraint variants of the Spherical Centroidal Voronoi Tessellation method.

Four-channel surface coil array for sequential CW-EPR image acquisition.

This article describes a four-channel surface coil array to increase the area of visualization for continuous-wave electron paramagnetic resonance (CW-EPR) imaging. A 776-MHz surface coil array was constructed with four independent surface coil resonators and three kinds of switches. Control circuits for switching the resonators were also built to sequentially perform EPR image acquisition for each resonator. The resonance frequencies of the resonators were shifted using PIN diode switches to decouple the inductively coupled coils. To investigate the area of visualization with the surface coil array, three-dimensional EPR imaging was performed using a glass cell phantom filled with a solution of nitroxyl radicals. The area of visualization obtained with the surface coil array was increased approximately 3.5-fold in comparison to that with a single surface coil resonator. Furthermore, to demonstrate the applicability of this surface coil array to animal imaging, three-dimensional EPR imaging was performed in a living mouse with an exogenously injected nitroxyl radical imaging agent.